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Archive for the ‘Proteomics’ Category

We found the amateur home video on protein blotting and western blot analysis posted on teachinhawaii’s YouTube channel. The video is a decent step-by-step demonstration of how to perform protein blotting and western blot analysis. It is appropriate for novice or first-time users. The video shows how to do protein blotting with Bio-Rad’s Mini-Trans Blot or Criterion Protein Blotting Systems. Of course, faster protein blotting can now be perfomed in under 3 minutes with Bio-Rad’s Trans-Blot Turbo Transfer System (as opposed to the 1-2 hours suggested in this video).

We are in the process of collecting protein blotting home videos. If you are aware of an interesting video, please let us know.

Here are some great application tips from Bio-Rad Laboratories for those researchers working with proteins:

Generally, the best method for keeping a protein in solution is to add any combination of nonionic detergents, zwitterionic detergents, and chaotropic agents to the sample mixture. Also use reducing agents such as DTT and DTE (less than 20 mM) to decrease disulfide bond formation between proteins.

When working with membrane or insoluble proteins, increase the amount of SDS in the equilibration and running buffers (up to 0.2%) to allow the proteins to effectively migrate out of the IPG strip.

To reduce the amount of SDS in samples generated by preparative SDS-PAGE, substitute the elution buffer with one that does not contain SDS.

Nucleic acid contamination is a common cause of horizontal gel streaking. Treat samples with nucleases to remove nucleic acids prior to isoelectric focusing.

Never heat samples in urea-containing buffers. The urea rapidly breaks down to carbamic acid and carbamylates the proteins, modifying their charge. Urea breakdown and subsequent protein carbamylation is the cause of charge trains on 2-D gels. A charge train is a series of spots on a 2-D gel that are of different pIs and the same size.

Two-dimensional (2-D) gel electrophoresis is a popular and proven separation technique for proteome analysis. The 2-D procedure is straightforward: Proteins are first separated according to their isoelectric point (pI) by isoelectric focusing (IEF) and then by their molecular weight by SDS-PAGE. For most researchers, 2-D gel electrophoresis is easy to learn, because advances in immobilized pH gradient (IPG) technology have eliminated the need for tricky and tedious IEF in ampholyte gel gradients. Nevertheless, problems with smearing, streaking, and poor resolution and reproducibility tend to leave researchers dissatisfied with the results of 2-D experiments. These common compalints are often due to improper sample preparation.

One of teh most undervalued aspects of the 2-D process, sample preparation prior to the first-dimension IEF separation contributes significantly to the overall reproducibility and accuracy of protein expression analysis. Some important considerations include:

Care must be taken to prevent protolysis during protein extraction, and proteins must be solubilized in a buffer that is compatible with IEF

Contaminants such as salts and detergents must be removed to ensure successful separation

Fractionation is essential to reduce protein sample complexity when analysis of subpopulations or low-abundance proteins is required

I was recently alerted to a new NIH funding opportunity described in this GenomeWeb News post which I thought would be of interest to the American Biotechnologist audience.

Funding Opportunity Announcement (FOA) Number PA-11-215 is a Small Business Innovation Research (STTR) grant for research involved in developing new technology for proteomics and glycomics. The funding opportunity was originally issued in April 2006 as PA-04-089 and has undergone several revisions in the intervening years.

Interestingly, despite major advances in the field of proteomics since 2006, there are many similarities in the language of the original announcement and the most recent version. In particular, I was struck by the comparability of the following two blurbs:

From 2006:

Technologies and methods remain largely inadequate to address the majority of meaningful biological problems, particularly with respect to quantitative and real time measurements.

From 2011:

Despite explosive growth in both academic and commercial efforts, concrete technical capabilities are far from adequate to realize this promise. Proteomics technologies and methods in the three broad, interacting domains of biology, analytical chemistry, and informatics are still largely inadequate to address the bulk of challenging biological problems.

I guess that one lesson to be learned from this comparison is that despite rapid techonological advances, lots of work remains to be done!

The NIH will provide up to $350,000 for Phase I and $600,000 for Phase II grants for projects addressing an important problem or a critical barrier to progress in the field of proteomics and glycomics that have commercial potential to lead to a marketable product, process or service.

The earliest submission date for the grant is July 5, 2011 and the grant expires on May 8, 2014. Interested applicants should visit the relevant NIH project announcement page.